Calcium ions govern many aspects of normal physiology and metabolism in all organisms and several pathological conditions involve aberrant calcium signalling. It is critical therefore that the roles of calcium ions in intracellular and inter-cellular regulation be understood. Calmodulin, a small highly conserved protein, mediates many of the intracellular responses to increased levels of calcium ions. Binding of calcium to the four calcium binding sites of calmodulin results in conformational changes which permit calmodulin to bind and activate a series of intracellular targets. We are using the model organism Drosophila melanogaster (a fruit fly) to study the function of calmodulin. All work to date indicates that the functions of calmodulin in Drosophila are very similar to its functions in mammals and thus our studies will be directly applicable to mammalian species. We are interested in the role of the individual calcium binding sites of calmodulin and have generated a series of mutants in which the calcium binding sites are incapacitated. Mutants with from one to four of the binding sites mutated have been generated. Physical studies such as NMR, X-ray crystallography, UV spectroscopy, Ca2+-binding and Ca2+-dissociation kinetics will be used to investigate the effects of these mutations on Ca2+-binding, Ca2+-induced conformational change and target binding and activation. A series of Ca2+-binding site mutants with a spectroscopic probe for study of conformational changes in the N-terminal region of calmodulin has also been generated. In addition, we will attempt to generate site-directed mutants in which calmodulin is trapped in the Ca2+-induced conformation. A major advantage of studying the organism Drosophila is that a genetic approach to studies of protein function can be applied. We have recently isolated mutations at the endogenous calmodulin gene of Drosophila. This puts us in the unique position of being able to analyze the effects of specific mutations to calmodulin in a living multicellular organism. Loss of calmodulin function in Drosophila results in embryonic death mainly as a result of defects in the nervous system. We will determine the extent to which mutants defective in one or more calcium binding sites can rescue these effects. The sophisticated genetics of Drosophila will also allow us to examine the effects of calmodulin loss at stages beyond embryogenesis in the individual tissues of the organism. We will perform this analysis and then examine the ability of our calcium binding site mutants to rescue the effects of calmodulin loss in these tissues.